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Team:StanfordBrownSpelman/Biodegradability
From 2014.igem.org
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Revision as of 05:53, 16 October 2014
Biodegradability
Approach & Methods
Methods here.
Figure 1. Figure caption here.
Results
Our goal for this project was not only to isolate biodegradation enzymes but also to control the release of these enzymes, so that our UAV would not degrade uncontrollably. In order to control the initiation of biodegradation, we first considered using a pressure sensor. This would allow the UAV to begin degrading after the impact of a crash. The 2008 Tokyo Tech iGem team found that the ptet promoter was pressure sensitive and increased its activity 3-fold after undergoing 30 MPa of pressure. We got their construct from the distribution kit and tested its functionality by monitoring GFP expression. We found that this promoter is always turned on even at very low pressures (The cells were bright green even with no pressure at all). When we introduced a repressor for the Ptet promoter, we found out that the repressor is too strong, and even at high pressures, the Ptet promoter will still be repressed. The photo and graphs below show our testing of the Ptet promoter in the presence and absence of the Tet repressor.
This graph shows that there was not a clear correlation between the amount of pressure applied on the ptet promoter to the amount of GFP expression: For instance, application of 14000rpm pressure on cells with ptet promoter for 30 seconds led to production of less fluorescence than application of 5000rpm pressure to the same batch of cells.When Tet Repressor was used, its effect was too strong and we could not find a repressor that could inhibit ptet just enough to control the biodegradation process
On top of testing the ptet promoter, we analyzed the impact a crash would have on our UAV by using a force plate and we found it unlikely that the impact of the crash would ever reach such a high pressure (see graph below).
JEANETTE OR JOVITA PLEASE INSERT THE PRESSURE/FORCE PLATE GRAPH HERE. IT IS NOT IN THE GOOGLE DRIVE SO I CAN'T DO IT.'
We next tried to initiate degradation using a light sensor, which would activate degradation in the darkness, allowing our UAV to have a flight time of one day. We planned to do this using the construct from the UT Austin and UCSF 2004 Coliroid project. However, the strain of E. coli (EnvZ) that we needed to work with to use this construct was resistant to all 4 of the main antibiotics we had in our lab, making the bacteria difficult and expensive to work with.
Finally, we decided to use quorum sensing as a means of creating a time delay for initiation of degradation. Two previous constructs, BBa_I13202 and BBa_T9002, when combined, create a quorum sensing cascade used to initiate expression of GFP. We ligated these two parts together to create our novel part, BBa_K1499500, which we used for our assays. We found that, in lac deficient cells, the quorum sensing construct can be initiated by induction with IPTG. After this induction, GFP expression increases with time, implying that the construct is doing its job. Our data for this assay is shown below.
'''ARYO PLEASE INSERT THE FLOW CYTOMETRY DATA HERE SINCE I DON'T HAVE IT.'''
Since we know that the quorum sensing construct is functional and inducible with IPTG. We can now work towards replacing the GFP gene with the genes for our degradation enzymes, allowing us to control degradation by applying IPTG at different time points.
In conjunction with working on controlling the initiation of degradation, we simultaneously worked with two degradation enzymes, esterase and cellulase. Both of these genes are isolated from the organism Neisseria sicca; the esterase is designed to de-acetylate cellulose acetate (our building material), and the cellulase breaks down the leftover cellulose into glucose monomers.
After successfully transforming the esterase gene into E. coli, and confirming via colony PCR, we grew up a large culture of transformed cells and used this to extract and purify the esterase protein. The protein gel confirmed that we had isolated the esterase protein, which is present near 43 kDa on the gel.
JEANETTE OR JOVITA PLEASE INSERT THE PRESSURE/FORCE PLATE GRAPH HERE. IT IS NOT IN THE GOOGLE DRIVE SO I CAN'T DO IT.'
We next tried to initiate degradation using a light sensor, which would activate degradation in the darkness, allowing our UAV to have a flight time of one day. We planned to do this using the construct from the UT Austin and UCSF 2004 Coliroid project. However, the strain of E. coli (EnvZ) that we needed to work with to use this construct was resistant to all 4 of the main antibiotics we had in our lab, making the bacteria difficult and expensive to work with. Finally, we decided to use quorum sensing as a means of creating a time delay for initiation of degradation. Two previous constructs, BBa_I13202 and BBa_T9002, when combined, create a quorum sensing cascade used to initiate expression of GFP. We ligated these two parts together to create our novel part, BBa_K1499500, which we used for our assays. We found that, in lac deficient cells, the quorum sensing construct can be initiated by induction with IPTG. After this induction, GFP expression increases with time, implying that the construct is doing its job. Our data for this assay is shown below.
'''ARYO PLEASE INSERT THE FLOW CYTOMETRY DATA HERE SINCE I DON'T HAVE IT.'''
Since we know that the quorum sensing construct is functional and inducible with IPTG. We can now work towards replacing the GFP gene with the genes for our degradation enzymes, allowing us to control degradation by applying IPTG at different time points.
In conjunction with working on controlling the initiation of degradation, we simultaneously worked with two degradation enzymes, esterase and cellulase. Both of these genes are isolated from the organism Neisseria sicca; the esterase is designed to de-acetylate cellulose acetate (our building material), and the cellulase breaks down the leftover cellulose into glucose monomers. After successfully transforming the esterase gene into E. coli, and confirming via colony PCR, we grew up a large culture of transformed cells and used this to extract and purify the esterase protein. The protein gel confirmed that we had isolated the esterase protein, which is present near 43 kDa on the gel.
Image description goes here.
INSERT GEL IMAGES FOR ESTERASE COLONY PCR AND PROTEIN GEL HERE.
Once we had isolated our protein we were able to do functional assays with the esterase enzyme. By using a cellulose-binding dye that selectively binds to cellulose and not cellulose acetate, we were able to test whether or not the esterase enzyme was effective in de-acetylating commercial grade cellulose acetate. We soaked the cellulose acetate in the esterase protein at its optimal temperature of 30ºC and tested with the stain at multiple points. The results of our assay (shown below) demonstrate that over time the protein was working to degrade the cellulose acetate, as the blue stain intensity increased over time.
THIS IS WHERE THE ESTERASE ASSAY IMAGE SHOULD BE BUT I CAN'T FIND IT...
We are currently working on functional assays of the cellulase gene, and have submitted it as a BioBrick (BBa_K1499501).
Once we had isolated our protein we were able to do functional assays with the esterase enzyme. By using a cellulose-binding dye that selectively binds to cellulose and not cellulose acetate, we were able to test whether or not the esterase enzyme was effective in de-acetylating commercial grade cellulose acetate. We soaked the cellulose acetate in the esterase protein at its optimal temperature of 30ºC and tested with the stain at multiple points. The results of our assay (shown below) demonstrate that over time the protein was working to degrade the cellulose acetate, as the blue stain intensity increased over time.
We are currently working on functional assays of the cellulase gene, and have submitted it as a BioBrick (BBa_K1499501).
